The long term objective of this application is to elucidate the protein factors that are involved in regulating stationary phase expressed genes in extremely halophilic members of the domain Archaea. This is of interest for two reasons. First, the transcription system in the haloarchaea is a minimalist version of the RNA polymerase II system found in the Eucarya. In addition to a single, multi-subunit RNA polymerase and a TATA box-like promoter, these organisms use only two of the factors involved for assembly of the basal transcription apparatus: homologs to eucaryal TATA-binding protein (TBP) and to transcription factor IIB (TFIIB). Second, the extreme halophiles are the only archaeons that possess multiple copies of TBP and TFIIB. Reeve et al. (1997) postulate that a combination of these factors may play a role in haloarchaeal gene regulation. To test this hypothesis, the genes that encode the various TBP and TFIIB factors present in Halobacterium spp. GN101 will be isolated using PCR products amplified with primers designed from the homologous genes in Hb. Salinarum NRC-1. These genes will be "knocked out" using a gene replacement protocol designed for extreme halophiles. The halR1 gene, which encodes the protein antibiotic halocin R1 and which is expressed in stationary phase, will be used as a "reporter" gene for assaying the effect of mutations in TBP and TFIIB genes. Similarily, there may be haloarchaeal analogs to the eucaryal TATA-binding associated factors (TAFs), although none have been found in any of the archaeal genomes so far sequenced. These trans-acting regulatory factors will be isolated by mutagenizing a halR1 mero-polyploid strain of GN101 and screening for mutants that have altered halocin R1 production. Finally, halocins themselves are fascinating proteins. For example, halocin H7 is effective in protecting the myocardium in dogs against deleterious effects of ischaemia and reperfusion (Alberola et. al, 1998).